Cardiac muscle function and manipulation

ABSTRACT

A method of causing cardiomyocyte growth and/or differentiation, the method comprising exposing a cardiomyocyte to neuregulin (NRG) thereby activating the MAP kinase pathway in the cardiomyocyte and causing growth and/or differentiation of the cardiomyocyte. Use of neuregulin, neuregulin polypeptide, neuregulin derivatives, or compounds which mimic the activities of neuregulins in the treatment or management of heart disease and heart failure in a mammal.

TECHNICAL FIELD

This invention relates to polypeptides which affect myocardial celldifferentiation and organisation of cardiac muscle contractile units,assay for identifying such polypeptides, and methods for improvingcardiac function by the administration of such polypeptides to patientswith heart disease.

BACKGROUND OF THE INVENTION

Heart failure affects 1.5% of populations, approximately three millionAmericans, developing at a rate of approximately 400,000 new cases peryear in USA. Current therapy for heart failure is primarily directed tousing angiotensin-converting enzyme (ACE) inhibitors and diuretics. ACEinhibitors appear to slow progress to end-stage heart failure; however,they are unable to relieve symptoms in more than 60% of heart failurepatients and reduce mortality of heart failure only by approximately15–20%. Heart transplantation is limited by the availability of donorhearts. With the exception of digoxin, the chronic administration ofpositive inotropic agents has not resulted in a useful drug withoutadverse side effects, including increased arrhythmias, or sudden death.These deficiencies in current therapy suggest the need for additionaltherapeutic approaches.

Growth of cardiac muscle cells switches from proliferation tohypertrophy during heart development. The former process ischaracterised by an increase in cardiac muscle cell number, and thelatter by an increase in cell size without DNA synthesis or celldivision. This switch is associated with terminal differentiation ofcardiac muscle cells and occurs gradually during heart development,starting during the late embryonic stages and ending a few weeks afterbirth. During this period, gene expression, particularly that involvingthe cell cycle and signalling, is reprogrammed. For example, expressionof a number of receptor protein tyrosine kinases and other cell cyclecomponents decreases. Cell phenotype is also changed as cell-celladhesions and contractile proteins are more organised in terminaldifferentiated myocardial cells.

Adult heart hypertrophy is an important adaptive physiological responseto increased demands for cardiac work or after a variety of pathologicalstimuli that lead to cardiac injury. Normal hypertrophic cells have alarge size with increased and well organised contractile units, as wellas strong cell-cell adhesions. Although pathologically hypertrophiccells also have large size and accumulation of proteins, they oftendisplay disorganisation of contractile proteins (disarray of sarcomericstructures) and poor cell-cell adhesions (disarray of myofibers). Thus,in pathological hypertrophy, the increase in size and accumulation ofcontractile proteins are associated with disorganised assembly ofsarcomeric structures and a lack of robust cell-cell interactions(Braunwald (1994) in Pathphysiology of Heart Failure. (Braunwald, ed.);Saunsers, Philadelphia; Vol. 14, pp 393–402).

The disarray of myofibers and sarcomeres are important features ofcardiomyopathy. The former is a disorder of cell-cell association, andthe latter is disorganisation of heart muscle contractile proteins. Theyare influenced by specific cell signals. Thus, a number of signals, likegrowth factors and hormones, alter cell adhesion and sarcomericstructure. Without these stimuli, cardiomyocytes display disarray of thecytoskeleton and sarcomeric structures, as well as disassociation ofcell-cell interactions. As cardiac muscle cell differentiation istightly associated with cardiac cell remodelling, adhesion andcontractile protein organisations, factors that stimulate myocardialcell differentiation may be critical for enhancing the assembly of adultcardiac muscle cell sarcomeric structures.

Studies in an in vitro model system of cardiac muscle cell have led tothe identification of a number of mechanical, hormonal, growth factor,and pathological stimuli which can activate several independentphenotype features of cardiac hypertrophy (Chien et al. (1991) FASEB J.5:3037–3046; Zhou et al., (1995) PNAS. USA, 92:7391–7395). Currently,there are at least three signal transduction pathways, involving bothras-, rho- and G_(q) protein-dependent downstream effectors implicatedin the activation of features of the hypertrophic response in these invitro model systems. While a great deal of progress has been made inuncovering the signalling pathways which activate the ventricular musclecell hypertrophic response, relatively little is known about themechanisms which specifically stimulate terminal differentiation ofcardiac muscle cells and the terminal differentiation-associatedassembly of contractile proteins. Compounds that could influence theseprocesses may be form a major new class of therapeutics for thetreatment of a variety of cardiac diseases.

Neuregulins, a family of EGF-like growth factors, activate ErbB receptortyrosine kinases that belong to the EGF receptor superfamily, and areinvolved in an array of biological responses: stimulation of breastcancer cell differentiation and secretion of milk proteins; induction ofneural crest cell differentiation to Schwann cells: stimulation ofskeletal muscle cell synthesis of acetylcholine receptors; and,promotion of myocardial cell survival and DNA synthesis. In vivo studiesof neuregulin gene-targeted homozygous mouse embryos with severe defectsin ventricular trabeculae formation and dorsal root ganglia developmentindicate that neuregulin is essential for heart and neural development.However, information on how neuregulin controls cell differentiation andits downstream signalling pathways is limited.

Within the heart, neuregulin and ErbB receptors are respectivelyexpressed in the endocardial lining and cardiac muscle layer in earlystages of development. Since these two layers are widely separated, theneuregulin ligand must transverse the space between the two cell layersto activate their cognate ErbB receptors. Activation of these receptorsin myocardial cells is necessary for promoting muscle cell growth ormigration toward the endocardium, which results in the formation offinger-like structures (ventricular trabeculae) between these twolayers. It is not clear previously if neuregulin stimulates myocardialcell differentiation.

The present inventor has now found that neuregulin and/or its cellularaction may be suitable for use in detection, diagnosis and treatment ofheart disease. Moreover, the inventor believes that potential beneficialeffects of neuregulin and/or its cellular action may be specific forheart muscle cells and not necessarily applicable to skeletal or smoothmuscle cells since 1) heart, skeletal and smooth muscle are bothembryological and functionally distinct; 2) factors involved in skeletalmuscle growth and differentiation, such as MyoD, play little or no rolein cardiac muscle growth and differentiation; 3) inactivation of thegenes for ErbB2 or 4 receptors or neuregulin produces major defects incardiac but not skeletal or smooth muscle development, 4) as shown here,the growth factor, insulin like growth factor-I (IGF-I) causes embryonicmyocyte proliferation but unlike neuregulin does not stimulatedifferentiation of these cells. By contrast, IGF-I but not neuregulin,has been shown to induce muscle hypertrophy.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that neuregulinenhances cardiac muscle cell differentiation and organisation ofsarcomeric and cytoskeleton structures, as well as cell-cell adhesion.Neuregulin, neuregulin polypeptide, neuregulin derivatives, or compoundswhich mimic the activities of neuregulins, fall within the scope of themethods of the present invention and are abbreviated hereinafter as NRG.

In a first aspect, the present invention consists in a method of causingcardiomyocyte growth and/or differentiation, the method comprisingexposing the cardiomyocyte to NRG thereby activating the MAP kinasepathway in the cardiomyocyte and causing growth and/or differentiationof the cardiomyocyte.

In a second aspect, the present invention consists in a method ofinducing remodelling of muscle cell sarcomeric and cytoskeletonstructures, or cell-cell adhesions, the method comprising treating thecells with neuregulin thereby activating the MAP kinase pathway in thecells and causing remodelling of the cell structures or the cell-celladhesions.

It will be appreciated that neuregulin may be provided directly to thecell or provided indirectly by causing neuregulin to be produced incells by inducing expression of the gene(s) involved in neuregulinproduction. The production may be in the same cell to which the methodis directed in an autocrine manner or by some other cell in a paracrinemanner.

In a third aspect, the present invention consists in a method ofidentifying polypeptides or compounds which stimulate cardiac musclecell differentiation, the method comprising contacting the cardiacmuscle with a test polypeptide or compound in the presence of an inducerof cardiac muscle cell proliferation in the form of neuregulin, andmeasuring the development of cardiac muscle cell differentiation.

The differentiation of cardiac muscle cells is preferably measured incells exposed to neuregulin or other test polypeptides, or to a mixtureof neuregulin with a test polypeptide. Differentiation of cardiac musclecell can be measured in a variety of ways, including by calculation ofincreases or decreases in DNA synthesis, analysis of the time-course ofphosphorylation of MAP kinases in cardiac muscle cells, evaluation ofthe expression of cell cycle inhibitor, p21^(CIP1), phenotypicorganisation of contractile units, accumulation of contractile units,phenotypic alteration of cytoskeleton actin fibers, and the phenotype ofcell-cell adhesions.

In one preferred embodiment of the method of identifying polypeptides orcompounds which stimulate cardiac muscle cell differentiation, cells areincubated with different concentrations of various peptides or compoundsand the effect of the test peptide or compound in differentconcentrations on cardiac muscle cell differentiation measured.

In another preferred embodiment of identifying polypeptides or compoundswhich induce cardiac muscle cell differentiation that dominates overthat of the putative inducer of cardiac muscle cell proliferation,insulin-like growth factor-1 (IGF-1), cells are incubated with IGF-1,with and without the test polypeptide or compound, and the ability ofthe test polypeptide or compound to inhibit IGF-1-mediated cardiacmuscle cell DNA synthesis, assembly of sarcomeric structures andcell-cell adhesions are measured.

In a further embodiment, the cells are incubated with phenylephrine (PE)with and without the test polypeptide or compound, and the ability ofthe test polypeptide or compound to augment PE-mediated cardiac musclecell differentiation is determined. A test polypeptide which stimulatescardiac muscle cell differentiation may stimulate the assembly ofsarcomeres and thus enhance heart function in a variety of ways,including by activating neuregulin-specific receptors, e.g., ErbB2,ERbB3 and ErbB4.

In a fourth aspect, the present invention consists in a method ofidentifying polypeptides or compounds which inhibit neuregulinstimulation of ventricular muscle cell differentiation, the methodcomprising contacting the ventricular muscle cell with the testpolypeptide or compound in the presence neuregulin and measuring anyinhibition of neuregulin stimulation of the ventricular muscle cell.

A compound may inhibit neuregulin stimulation of ventricular muscle celldifferentiation by blocking, suppressing, reversing, or antagonising theaction of neuregulin. In one embodiment, the measurement is by detectingDNA synthesis of ventricular muscle cells.

In a fifth aspect, the present invention consists in a therapeuticmethod of treating or preventing disassociation of cardiac musclecell-cell adhesion and/or the disarray of sarcomeric structures in amammal, the method comprising administering to the mammal atherapeutically effective amount of a neuregulin or its derivatives.

In one preferred embodiment, the therapeutic method is directed totreating heart failure resulting from disassociation of cardiac musclecell-cell adhesion and/or the disarray of sarcomeric structures in themammal.

In a sixth aspect, the present invention consists in a method ofpreventing or lowering the incidence of heart disease in a mammal, themethod comprising preventing or lowering the interference or effects ofpolypeptides or compounds on the action of neuregulin and its receptors,ErbBs, that produces heart failure.

In another embodiment, a therapeutic agent which mimics the effects ofneuregulin is used to treat or prevent PE, or IGF-1-mediated cardiacmuscle cell dysfunction.

In an seventh aspect, the present invention consists in a method ofdetermining predisposition to heart disease or heart failure in asubject, the method comprising testing cardiac or related cells of thesubject for the ability to express and/or produce normal or adequatelevels of neuregulin or its cognate ErbB receptors. The inability toexpress and/or produce normal or adequate levels of neuregulin beingindicative of predisposition to heart disease or heart failure.

In a eighth aspect, the present invention consists in the use ofneuregulin, neuregulin polypeptide, neuregulin derivatives, or compoundswhich mimic the activities of neuregulins in the treatment or managementof heart disease and heart failure.

In a ninth aspect, the present invention consists in the use ofneuregulin, neuregulin polypeptide, neuregulin derivatives, or compoundswhich mimic the activities of neuregulins in the manufacture of amedicament for the treatment or management of heart disease and heartfailure.

By using primary cultured myocardial cells as a model system, thepresent inventor evaluated neuregulin signalling in cardiac myocytedifferentiation, maturation and assembly or maintenance of sarcomericand cytoskeleton structures. To assay the neuregulin effect on cellsignalling, embryonic cardiac muscle cells were incubated withrecombinantly purified human neuregulin ligand (rhNRGβ2). Neuregulin at10⁻⁸M resulted in sustained activation of MAP kinases for at least 21hours, whereas only transient activation was observed with a lowerconcentration (10⁻¹⁰M) of rhNRGβ2. Expression of the Cdk inhibitor,p21^(CIP1), was enhanced by the 10⁻⁸M, but not the 10⁻¹⁰M concentrationof the ligand. The higher ligand concentration, concomitant with thisincrease in p21^(CIP1) expression, resulted in a decrease in DNAsynthesis, that was associated with terminal differentiation, whereas anincrease in DNA synthesis and continued proliferation was observed withthe lower dose. Furthermore, when neuregulin was mixed with IGF-1,rhNRGβ2 at either concentrations (10⁻⁸M, or 10⁻¹⁰M) did not show anegative effect on the DNA synthesis and significantly blockedIGF-1-stimulated cardiomyocyte proliferation. To further evaluate theNRG-stimulated myocardial cell differentiation, sarcomeric andcytoskeleton structures of cultured neonatal rat cardiac muscle cellswere examined by Phalloidin staining and immunofluorescent staining withanti-α-actinin antibody. rhNRGβ2 dramatically improved sarcomeric andcytoskeleton structures, as well as cell-cell adhesions. Such an effectwas not found from the cells stimulated with either insulin IGF-1 or PE.When rhNRGβ2 was mixed with either IGF-1 or PE, rhNRGβ2 improved thecell structures. The 10⁻⁸M concentration of rhNRGβ2 showed maximaleffect on improvements of sarcomeres and cell-cell adhesions. Inaddition, neuregulin overrode the negative regulation of MHC-αexpression mediated by PE stimulation. These findings indicate that NRGfunction through two distinct pathways: one activated at lower ligandconcentrations results in cardiomyocyte growth, whereas the other,activated with higher concentrations, is mediated by sustainedactivation of the MAP kinase pathway and results in terminaldifferentiation and maturation.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element, integeror step, or group of elements, integers or steps, but not the exclusionof any other element, integer or step, or group of elements, integers orsteps.

In order that the present invention may be more clearly understood,preferred forms will be described with reference to the followingexamples and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Growth factor-stimulated DNA synthesis. DNA synthesis ([³H]thymidine incorporation) by cultured embryonic mouse cardiomyocytes inresponse to 20 hr of treatment with the indicated concentrations ofvehicle (purified Flag-peptide) (open square), recombinant human NRGβ2(rhNRGβ2) (closed triangle) or insulin-like growth factor-I (IGF-I)(open circle). Data shown are the mean±S.E of five determinations witheach treatment and at each concentration. All rhNRGβ2 reponses aresignificantly greater than control (P<0.001) except at 10⁻⁷M, and therhNRGβ2 responses to concentration ³ 10⁻⁹M are significant than therespective IGF-I responses (P<0.01).

FIG. 2. NRG-mediated ErbB receptor phosphorylation. (a) Serum-starvedcardiomyocytes were stimulated with vehicle (0) or rhNRGβ2 at aconcentration of either 10⁻¹⁰M or 10⁻⁸M for the times indicated.Phosphorylation of ErbB receptors was then determined as described inMethods using an anti-phosphotyrosine antibody (RC20H). Fold increasesin immunoblot intensities are shown, which were normalised for proteinload based on the intensities of simultaneously determined immunoblotsof ErbB2 shown below the phosphotyrosine species. (b) Phosphorylation ofimmunoprecipitated ErbB2 (top panel) or ErbB4 (bottom panel) resultingfrom the stimulation of embryonic cardiomyocytes for 5 min with 10⁻¹⁰Mor 10⁻⁸M rhNRGβ2. Studies were performed as detailed in Methods and theimmunoprecipitated products evaluated by immuno-blotting withanti-phosphotyrosine, anti-ErbB2 or anti-ErbB4 antibodies. Fold changesin immunoblot intensities are shown, which were normalised for proteinloading based on the intensities of simultaneously determinedimmunoblots of ErbB2 or ErbB4 shown below the phosphotyrosine species.

FIG. 3. NRG or IGF-I stimulated MAP kinase activation. (a) MAP kinasephosphorylation resulting from embryonic cardiomyocyte stimulation withrhNRGβ2 (10⁻¹⁰M or 10⁻⁸M) for the times shown. After treatment withrhNRGβ2, cell extracts were prepared and evaluated for MAP kinasephosphorylation using an anti-phospho MAP kinase antibody, as describedin Methods. Fold changes in immunoblot intensities are shown below thephosphotyrosine species. To control for protein loading, cell extractswere simultaneously evaluated for ErbB2 expression by immunoblotanalysis using an anti-ErbB2 antibody. (b) MAP kinases catalyticactivity was determined as described in Methods using extracts preparedfrom cardiomyocytes treated with rhNRGβ2 (10⁻¹⁰M or 10⁻⁸M) for the timesindicated, and shown as fold increase over the basal level activity ofcells which were not stimulated with rhNRGβ2. Values of the fold shownas the means±SE of five determinations with each treatment and at eachconcentration. (c) MAP kinase phosphorylation resulting from IGF-I(10⁻⁹M) stimulation of embryonic cardiomyocytes for the times indicated,was determined as in (a).

FIG. 4. Effects of NRG on IGF-I stimulated DNA synthesis and MAP kinasephosphorylation. (a) DNA synthesis ([³H] thymidine incorporation) wasexamined in cultured cells stimulated with a maximal concentration ofIGF-I (10⁻⁹M) in the absence or presence of rhNRGβ2 at concentrations ofeither 10⁻¹⁰M or 10⁻⁸M for 20 hrs. Bars show the mean values of datafrom five samples±1S.E (error bars). Similar results were obtained fromthree independent experiments. Significant difference (***, P<0.001)from the control are indicated. (b) Time course of MAPK phosphorylationin embryonic cardiac muscle cells in response to a mixture of 10⁻⁹MIGF-I and 10⁻⁸M rhNRGβ2 was determined by immunoblotting using aspecific anti-phospho-MAPK antibody. ErbB2 expression was evaluatedsimultaneously to control for protein loading.

FIG. 5. NRG-mediated induction of p21^(CIP1) expression. (a) p21^(CIP1)expression in cultured cardiac muscle cells stimulated either with10⁻¹⁰M or 10⁻⁸M rhNRGβ2 in the absence or presence of serum (5% of FBS)for 24 hrs; or (b) with 10⁻¹⁰M or 10⁻⁸M IGF-I. After the varioustreatments, p21^(CIP1) expression was evaluated by immunoblot analysisusing an anti-p21^(CIP1) antibody. ErbB2 expression was evaluatedsimultaneously to control for protein loading. Fold changes inp21^(CIP1) expression, normalised for protein loading, are shown. (c)Effect of the MEK inhibitor (PD98059) (50 μM) on rhNRGβ2 (either at10⁻¹⁰M or 10⁻⁸M)-mediated stimulation of p21^(CIP1) expression incultured embryonic cardiac muscle cells in the absence of serum.p21^(CIP1) was detected by immunoblot analysis using an anti-p21^(CIP1)antibody. Fold changes in p21^(CIP1) expression, normalised for proteinloading, are shown. (d) Effects of PD98059 on the inhibition of NRG- orIGF-I-activated MAP kinase activities were monitored by a measurement ofp42/44 MAP kinase phosphorylation after cells were stimulated with NRGor IGF-I for 5 min. p42/44 MAP kinase phosphorylation was evaluated byimmunoblot analysis using anti-phospho-p42/44 or anti-p42/44 MAP kinaseantibodies. The same amount of whole cell extract (20 μg protein) wasloaded, and normalised for p42/44 MAP kinase expression, using ananti-p42/44 MAP kinase antibody.

FIG. 6. Effects of NRG on cardiac sarcomere assembly and myosin heavychains expression. (a) E12.5 mouse cardiac muscle cells were cultured inserum-free medium (control) or stimulated with 10⁻¹⁰ or 10⁻⁸M rhNRGβ2(NRG) for 48 hrs. Cells were then stained with phalloidin (left panels)or evaluated for anti-α-actinin immunoflorescency (right panels). (b)Sarcomeric myosin heavy chain and α-actin expression inrhNRGβ2-stimulated embryonic mouse cardiac muscle cells were evaluatedby immunoblot analysis using an anti-sarcomeric myosin heavy chainantibody (MF20) or an anti-α-actin antibody. The same amount of wholecell extracts (20 μg proteins) was loaded into each lane for SDS-PAGEfractionation.

MODES FOR CARRYING OUT THE INVENTION

Utilising an in vitro system of cardiac muscle cell differentiation, arole for neuregulin in stimulating the activation of the differentiationresponse in comparison with two well-defined hormonal and growth factorstimuli, α₁-adrenergic agonists and IGF-1 has been demonstrated. Thepresent inventor has demonstrated that neuregulin differentiationpathways exist within cardiac muscle cells, and that neuregulinpolypeptides can activate these pathways. Since cardiac muscle celldifferentiation includes the processes of organisation of sarcomericstructures and cell-cell adhesions, the invention, thus, provides auseful method for the treatment and prevention of cardiac muscle cellwith disorganisation of the sarcomeric structures and cell-celladhesions, and the enhancement of heart function in cardiomyopathy, andfor identifying polypeptides or compounds which activate cardiac muscledifferentiation pathways.

Before the methods of the invention are described, it is to beunderstood that this invention is not limited to the particular methodsdescribed. The terminology used herein is for the purpose of describingparticular embodiments only.

As used in this specification, the singular forms “a”, “an”, and “the”include plural references unless the context clearly dictates otherwise.Thus, for example, references to “neuregulin” or “a neuregulin peptide”includes mixtures of such neuregulins, neuregulin isoforms, and/orneuregulin-like polypeptides. Reference to “the formulation” or “themethod” includes one or more formulations, methods, and/or steps of thetype described herein and/or which will become apparent to those personsskilled in the art upon reading this disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of disclosing anddescribing material for which the reference was cited in connectionwith.

Definitions

“Neuregulin or neuregulin analogs” are molecules that can activateErbB2/ErbB4 or ErbB2/ErbB3 heterodimer protein tyrosine kinases, such asall neuregulin isoforms, neuregulin EGF domain alone, neuregulinmutants, and any kind of neuregulin-like gene products that alsoactivate the above receptors. The “neuregulin” used in this invention isthe following polypeptide which is a fragment of human neuregulin β2isoform containing the EGF-like domain, the receptor binding domain.

The cDNA sequence:

AGCCATCTTGTAAATGTGCGGAGAAGGAGAAAACTTTCTGTGTGAATGGAGGGGAGTGCTTCATGGTGAAAGACCTTTCAAACCCCTCGAGATACTTGT GAGGAGCTGTACCAG (SEQID NO:1)

The amino acid sequence encoded by the above DNA sequence:

SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVM ASFYKAEELYQ (SEQ IDNO:2).

“Cardiac muscle cell differentiation” is a condition characterised bythe decrease in DNA synthesis by more than 10%, inhibition of otherfactor-stimulated DNA synthesis more than 10%, well organised sarcomericstructures and cell-cell adhesions, sustained activation of MAP kinases,and enhanced expression of p21^(CIP1).

“Organised, or enhanced organisation of sarcomeres or sarcomericstructures” is a condition characterised by the straight array ofcontractile proteins revealed by immunofluorescent staining of α-actininin cardiac muscle cells. The straight array of α-actinin proteins incells can be distinguished by microscopy and its connected photographyas exampled in Figures of this specification.

“Disorganised or disarray of sarcomeres or sarcomeric structures” is theopposite meaning of the above definitions.

“Organised, or enhanced organisation of cytoskeleton structures” is acondition characterised by the straight actin fibers revealed byphalloidin staining of cardiac muscle cells. The straight actin fibersin cells can be distinguished by microscopy and its connectedphotography as exampled in Figures of this specification.

“Disorganised or disarray of cytoskeleton structures” is the oppositemeaning of the above definitions.

“Sustained activation of MAP kinases” is that phosphorylated state ofMAP kinases, p42/44, is maintained for at least 21 hr in cells.

“Enhanced expression of p21^(CIP1)” is that expression of p21^(CIP1) isincreased at least 50% that is maintained for at least 24 hr in cells.

“The treatment of heart diseases” includes all suitable kinds ofmethods, such as vein injection of the neuregulin polypeptide, and genetherapy methods, in which heart or other cells were forced to contain agene encoding neuregulin or derivatives for the treatment of heartdiseases. For example, Adenovirus or Adeno-Associated-Virus can be usedas a carrier to deliver neuregulin gene into infected heart or othercells. The infected cell can then express and secret neuregulinpolypeptide to activate ErbBs on cardiac muscle cells.

“Ventricular muscle cell hypertrophy” is a condition characterised by anincrease in the size of individual ventricular muscle cells, theincrease in cell size being sufficient to result in a clinical diagnosisof the patient or sufficient as to allow the cells to be determined aslarger (e.g., 2-fold or more larger than non-hypertrophic cells). It maybe accompanied by accumulation of contractile proteins within theindividual cardiac cells and activation of embryonic gene expression.

In vitro and in vivo methods for determining the presence of ventricularmuscle cell hypertrophy are known. In vitro assays for ventricularmuscle cell hypertrophy include those methods described herein, e.g.,increased cell size and increased expression of atrial natriureticfactor (AND). Changes in cell size are used in a scoring system todetermine the extent of hypertrophy. These changes can be viewed with aninverted phase microscope, and the degree of hypertrophy scored with anarbitrary scale of 7 to 0, with 7 being fully hypertrophied cells, and 3being non-stimulated cells. The 3 and 7 states may be seen in Simpson etal. (1982) Circulation Res. 51: 787–801, FIG. 2, A and B, respectively.The correlation between hypertrophy score and cell surface area (μm²)has been determined to be linear (correlation coefficient=0.99). Inphenylephrine-induced hypertrophy, non-exposed (normal) cells have ahypertrophy score of 3 and a surface area/cell of 581 μm² and fullyhypertrophied cells have a hypertrophy score of 7 and a surfacearea/cell of 1811 μm², or approximately 200% of normal. Cells with ahypertrophy score of 4 have a surface area/cell of 771 μm², orapproximately 30% greater size than non-exposed cells; cells with ahypertrophy score of 5 have a surface area/cell of 1109 μm², orapproximately 90% greater size than non-exposed cells; and cells with ahypertrophy score of 6 have a surface area/cell of 1366 μm², orapproximately 135% greater size than non-exposed cells. The presence ofventricular muscle cell hypertrophy preferably includes cells exhibitingan increased size of about 15% (hypertrophy score 3.5) or more. Inducersof hypertrophy vary in their ability to induce a maximal hypertrophicresponse as scored by the above-described assay. For example, themaximal increase in cell size induced by endothelin is approximately ahypertrophy score of 5.

“Suppression” of ventricular muscle cell hypertrophy means a reductionin one of the parameters indicating hypertrophy relative to thehypertrophic condition, or a prevention of an increase in one of theparameters indicating hypertrophy relative to the normal conditions. Forexample, suppression of ventricular muscle cell hypertrophy can bemeasured as a reduction in cell size relative to the hypertrophiccondition Suppression of ventricular muscle cell hypertrophy means adecrease of cell size of 10% or greater relative to that observed in thehypertrophic condition. More preferably, suppression of hypertrophymeans a decrease in cell size of 30% or greater; most preferably,suppression of hypertrophy means a decrease of cell size of 50% or more.Relative to the hypertrophy score assay when phenylephrine is used asthe inducing agent, these decreases would correlate with hypertrophyscores of about 6.5 or less, 5.0–5.5, and 4.0–5.0, respectively. When adifferent agent is used as the inducing agent, suppression is measurerelative to the maximum cell size (or hypertrophic score) measured inthe presence of that inducer.

Prevention of ventricular muscle cell hypertrophy is determined bypreventing an increase in cell size relative to normal cells, in thepresence of a concentration of inducer sufficient to fully inducehypertrophy. For example, prevention of hypertrophy means a cell sizeincrease less than 200% greater than non-induced cells in the presenceof maximally-stimulating concentration of inducer. More preferably,prevention of hypertrophy means a cell size increase less than 135%greater than non-induced cells; and most preferably, prevention ofhypertrophy means a cell size increase less than 90% greater thannon-induced cells. Relative to the hypertrophy score assay whenphenylephrine is used as the inducing agent, prevention of hypertrophyin the presence of a maximally-stimulating concentration ofphenylephrine means a hypertrophic score of about 6.0–6.5, 5.0–5.5, and4.0–4.5, respectively.

In vivo determination of hypertrophy include measurement ofcardiovascular parameters such as blood pressure, heart rate, systemicvascular resistance, contractility, force of heart beat, concentric ordilated hypertrophy, left ventricular systolic pressure, leftventricular mean pressure, left ventricular end-diastolic pressure,cardiac output, stroke index, histological parameters, and ventricularsize and wall thickness. Animal models available for determination ofdevelopment and suppression of ventricular muscle cell hypertrophy invivo include the pressure-overload mouse model, RV murine dysfunctionalmodel, transgenic mouse model, and post-myocardial infarction rat model.Medical methods for assessing the presence, development, and suppressionof ventricular muscle cell hypertrophy in human patients are known, andinclude, for example, measurements of diastolic and systolic parameters,estimates of ventricular mass, and pulmonary vein flows.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacological and/or physiologicaleffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, particularly a human, andincludes:

preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;

inhibiting the disease. i.e., arresting its development; or

relieving the disease, i.e., causing regression of the disease.

The invention is directed to treating patients with or at risk fordevelopment of heart disease and related conditions, e.g., heartfailure. More specifically, “treatment” is intended to mean providing atherapeutically detectable and beneficial effect on a patient sufferingfrom heart disease.

By the term “heart failure” is meant an abnormality of cardiac functionwhere the heart does not pump blood at the rate needed for therequirements of metabolising tissues. Heart failure includes a widerange of disease states such as congestive heart failure, myocardialinfarction, tachyarrythmia, familial hypertrophic cardiomyopathy,ischaemic heart disease, idiopathic dilated cardiomyopathy, andmyocarditis. The heart failure can be caused by any number of factors,including ischaemic, congenital, rheumatic, or idiopathic forms. Chroniccardiac hypertrophy is a significantly diseased state which is aprecursor to congestive heart failure and cardiac arrest.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) hypertrophy. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein which the disorder is to be prevented. The hypertrophy may be fromany cause which is responsive to retinoic acid, including congenital,viral, idiopathic, cardiotrophic, or myotrophic causes, or as a resultof ischaemia or ischaemic insults such as myocardial infarction.Typically, the treatment is performed to stop or slow the progression ofhypertrophy, especially after heart damage, such as from ischaemia, hasoccurred. Preferably, for treatment of myocardial infarctions, theagent(s) is given immediately after the myocardial infarction, toprevent or lessen hypertrophy.

The terms “synergistic, “synergistic effect” and like are used herein todescribe improved treatment effects obtained by combining one or moretherapeutic agents with one or more retinoic acid compounds. Although asynergistic effect in some fields is meant an effect which is more thanadditive (e.g., 1+1=3), in the field of medical therapy an additive(1+1=2) or less than additive (1+1=1.6) effect may be synergistic. Forexample, if each of two drugs were to inhibit the development ofventricular muscle cell hypertrophy by 50% if given individually, itwould not be expected that the two drugs would be combined to completelystop the development of ventricular muscle cell hypertrophy. In manyinstances, due to unacceptable side effects, the two drugs cannot beadministered together. In other instances, the drugs counteract eachother and slow the development of ventricular muscle cell hypertrophy byless than 50% when administered together. Thus, a synergistic effect issaid to be obtained if the two drugs slow the development of ventricularmuscle cell hypertrophy by more than 50% while not causing anunacceptable increase in adverse side effects.

Materials and Methods

Reagents and Antibodies

The following antibodies and reagents were used: IGF (Boehringer);collagenase (Worthington); pancreatin (Gibco BRL); MEK1 (MAPKK)inhibitor (PD98059) (New England); [methyl-³H]thymidine (Amersham);monoclonal anti-erbB2 antibody (Novocastra); monoclonal IgG_(2b)p21^(CIP1) (F-5) (Santa Cruz); monoclonal anti-phospho-tyrosine horseradish peroxidase (HRPO)-conjugated antibody, RC20 (TransductionLaboratories); monoclonal anti-α-sacromeric actin antibody (clone 5c5),HRPO-conjugated anti-rabbit Ig, and anti-mouse Ig (Sigma); PhosphoPlus®p44/42 MAP kinase (Thr202/Tyr204) antibody kit (New England); anti-FLAG®M1 affinity gel and anti-FLAG M2monoclonal antibody (Eastman Kodak) mAbMF20 to sarcomeric myosin heavy chain (kindly provided by R. P. Harvey,Victor Chang Cardiac Research Institute); anti-sarcomeric α-actinantibody (Sigma).

Recombinant Human NRGβ2 Expression and Purification

A cDNA encoding the EGF-like domain of human NRGβ2 isoform (rhNRGβ2),residues 177–237, was inserted into the pFLAG1 expression vector (IBI)(a gift from Dr. Rodney J. Fiddes, Co-operative Research Centre forBiopharmaceutical Research, Australia). rhNRGβ2 with a FLAG-peptideattached at its N-terminus, was expressed in the periplasmic space of E.coli DH5α, and purified by affinity chromatography using anti-FLAG M1monoclonal antibody according to the manufacturer's instructions. Thepurity of rhNRGβ2 was more than 90% as evidenced by SDS-PAGE separationand Coomassie Blue staining of purified protein samples. Theconcentration of purified proteins was determined using a Bio-Radprotein assay kit. Activity of the purified proteins was assayed bystimulation of MCF-7 breast cancer cell ErbB receptors with variousligand doses. This revealed increased ErbB receptor phosphorylation withincreasing ligand concentration (10⁻¹²M to 10⁻⁸M).

Primary Cultures of Mouse Cardiac Myocytes

Mouse embryos (E11.5–12.5) were used to prepare primary cardiacmyocytes. Heart tissue was isolated aseptically from embryos. Myocardialcells were isolated by collagenase digestion and separated fromnon-cardiomyocytes by preattachments on culture dishes that wasperformed three times. Cells were then cultured as described previously.Using this method, it was routinely possible to obtained primarycultures with >90% myocytes.

ErbB and MAP Kinase Phosphorylation, and MAP Kinase Activity

Embryonic myocardial cells were cultured in serum-free medium for atleast 24 hrs and then stimulated with rhNRGβ2 or IGF-I for varioustimes. Stimulation was terminated by washing cells rapidly with coldPBS. To block the MAP kinase activation, the MEK inhibitor, PD98059, wasadded to the medium 30 mins prior to the adminstration of rhNRGβ2 orIGF-I. Cells were then harvested as previously described for Westernblot analysis with HRPO-conjugated monoclonal antibody RC20H (1:2,000)for detection of phosphorylated ErbB receptors, or a phospho-specificp42/p44 MAP kinase antibody (dilution ratio 1:1,000) for detection ofphosphorylated MAP kinases. The same amount of cell extract protein wasloaded into each lane and separated by SDS-PAGE. Immunobloting with ananti-ErbB receptor or anti-p42/44 MAP kinase antibodies was also used tonormalise for protein loading. MAP-kinase (p42/p44) activity wasmeasured using a p42/44 MAP kinase enzyme assay kit (RPN84; Amersham,Bucks., U.K.) according to the manufacturer's instructions.

Detection of p21^(CIP1) Protein

Embryonic myocardial cells cultured in serum-free or 5% FBS medium werestimulated with various concentrations of rhNRGβ2 with or without theMEK inhibitor, PD98059, for 24 or 48 hrs, harvested as described aboveand subjected to immunoblot analysis using an anti-p21^(CIP1) antibody(1:100). The same amount of protein was loaded into each well of a SDSpolyacrylamide gel. After immunobloting, the membrane was stripped andprobed further with an antibody to the ErbB2 receptor for normalisationof protein loading.

Thymidine Incorporation

Embryonic myocardial cells were cultured with rhNRGβ2 or IGF-Icontaining serum-free DMEM for 20 hrs. [methyl-³H] Thymidine (0.5μCi/well) was added and cells were then cultured for a further 12 hrs.After rinsing twice with cold PBS, once with ice-cold 10%trichloroacetic acid, and then five times with ice-cold PBS, the cellswere dissolved in 100 μl of 1% SDS, and counted in a liquidscintillation counter.

Immunofluorescent and Phalloidin Staining

Myocardial cells were plated in 2-well Novex plates (Nunc), and culturedwith or without rhNRGβ2 in serum-free DMEM medium for 24–48 hour. Afterrinsing the cells with PBS, they were fixed with 4% paraformaldehyde and0.1% Triton X-100 at room temperature for 30 minutes. The fixed cellswere then blocked with 5% skim milk in PBS for 1 hr, followed byincubation with an anti-α-actinin monoclonal antibody (Sigma), for 45minutes at room temperature. After washing, anti-mouse IgG conjugatedwith FITC (Sigma) was added and the cells were incubated for anotherhalf hour. For phalloidin staining, cells were fixed with 4% formadehydefor 1 hr., washed, and stained with phalloidin buffer (100 μl PBS, 10 ulrhodamine phalloidin (6.6 μM in MeOH)) for 1 hr. After PBS washing,cells were mounted with 1% p-phenylenediamine (1 mg/ml, Sigma) inglycerol, and then covered and sealed. Cells were examined using a UVfluorescent microscope and photographed with a 40× power objective.

All of the above assays were repeated at least three times for eachexperiments. Data for DNA synthesis and MAP kinase activity arepresented as the mean±S.E of five replicate samples. Statisticalsignificance was determined by ANOVA using the SAS statistical packagewith P<0.05 being considered significant. Immunoblots were quantitatedby densitometry analysis with the intensity of the evaluated proteinbands being shown below the blots as the fold changes over control (seeFigures).

Results

NRG Regulates Embryonic Myocardial Cell DNA Synthesis

DNA synthesis in primary embryonic mouse cardiomyocytes (E11.5–12.5) wasevaluated to investigate their growth response to NRG followingstimulation with rhNRGβ2. As shown in FIG. 1, rhNRGβ2 at a concentrationof 10⁻¹⁰M produced an approximately 2-fold increase in the DNAsynthesis. However, DNA synthesis decreased with ligandconcentrations>10⁻¹⁰M. In contrast to the response to rhNRGβ2,myocardial cells showed only a proliferative response to recombinanthuman insulin-like growth factor I (IGF-I), in concentrations rangingfrom 10⁻¹¹M to 10⁻⁷M. Inhibition of DNA synthesis by the higherconcentrations of NRG was not due to E. coli proteins contaminating thebacterially-expressed rhNRGβ2, since proteins purified from bacteriatransformed with FLAG-vector alone did not inhibit DNA synthesis.Moreover, to avoid possible effects of E. coli proteins, bothcommercially obtained IGF-I and purified rhNRGβ2 were disolved ordiluted with anti-FLAG-protein preparations (10⁻⁸M of FLAG peptide).These reagents showed identical activities to those prepared with PBS instimulating myocardial cell DNA synthesis.

NRG Activates Embryonic Myocardial Cell ErbB Receptors

Of the four members of the ErbB receptor family (ErbB1–4), ErbB2 andErbB4 are most abundantly expressed in cardiac myocytes. Phosphorylationof ErbB2 and ErbB4 receptors was evaluated by Western blot analysis ofcell lysates, following stimulation with either 10⁻⁸M or 10⁻¹⁰M rhNRGβ2.As shown in FIG. 2 a, a higher level of phosphorylated 180–185 kDaproteins corresponding to ErbB2/ErbB4 receptors, was evident with thehigher concentration of NRG. The levels of phosphorylation graduallydecreased with time. The concentration dependence of p180–185 proteinphosphorylation corresponded to that for the decrease in DNA synthesiswith rhNRGβ2 treatment (FIG. 1). ErbB2 and ErbB4 receptors were alsoimmunoprecipitated using anti-ErbB2 or ErbB4 antibodies, and examined byWestern blotting with anti-phospho-tyrosine antibodies. As shown in FIG.2 b, phosphorylation of both receptors was dependent on rhNRGβ2concentrations. Although ErbB2 and ErbB4 phosphorylation levels differedslightly between experiments, the relative phosphorylation differencebetween high and low concentrations of rhNRGβ2 persisted.

NRG Concentration-Dependent Activation of MAP Kinases

Activation of the ErbB receptor family initiates a cascade of molecularinteractions, ultimately resulting in the stimulation of MAP kinases.The duration of MAP kinase activation is critical for cell-fatedecisions. Therefore, the present inventor investigated the time courseof MAP kinase phosphorylation after either 10⁻⁸M or 10⁻¹⁰M rhNRGβ2treatment, using a specific-phospho-MAP kinase antibody, whichrecognises phosphorylated p42/p44 MAP kinases. As shown in FIG. 3 a,phosphorylation of p42/p44 MAP kinases was sustained for at least 21hours with the higher dose of rhNRGβ2. MAP kinase activation wastransient at the lower ligand concentration, and fell to the basal levelin less than three hours. As shown in FIG. 3 b, MAP kinase catalyticactivity paralleled these changes in phosphorylation. Thus, MAP kinaseactivity was sustained for at least 21 hours in cells stimulated with10⁻⁸M rhNRGβ2, but was only transient in cells treated with 10⁻¹⁰MrhNRGβ2. In contrast to these NRG responses, MAP kinase phosphorylationwas transient both with low (10⁻⁹M) (FIG. 3 c) and with highconcentrations (10⁻⁸M or 10⁻⁷M) of IGF-I.

Effect of NRG on IGF-I-Stimulated Myocardial Cell Proliferation

Since myocardial cells are exposed to multiple peptide hormones andgrowth factors in vivo, the present inventor investigated if the growthinhibitory effects of a high concentration of NRG could oppose theproliferative response of other growth factors. This was achieved byevaluating the effects of both rhNRGβ2 and IGF-I on cardiac myocyte DNAsynthesis. As shown in FIG. 4 a, a 10⁻¹⁰M concentration of NRG hadlittle effect on IGF-I (10⁻⁹M)-stimulated DNA synthesis. However, the10⁻⁸M concentration significantly blocked the IGF-I response. Thisindicated that a specific intracellular pathway was activated by thehigher concentration of NRG. Interestingly, no additive effect wasobserved when both IGF-I and the lower concentration of NRG were appliedto cells, indicating that the 10⁻⁹M concentration of IGF-I may alreadybe maximal. That the pathway(s) activated by the higher concentration ofNRG may be dominant over that activated by IGF-I was further supportedby the observation that the combination of IGF-I (10⁻⁹M) and rhNRGβ2(10⁻⁸M) resulted in sustained MAP kinase phosphorylation (compare FIG. 4b and FIG. 3 c)

NRG and p21^(CIP1) Expression

Since sustained activation of MAP kinase is directly related to theexpression of p21^(CIP1) in other types of cells, ³¹ and accumulation ofp21^(CIP1) leads to cell cycle arrest at the G1 phase,^(32, 33) it wasasked if the sustained activation of MAP kinases leads to a higher levelof p21^(CIP1) expression in embryonic cardiac muscle cells. As shown inFIG. 5 a, an increase in p21^(CIP1) expression was observed only withthe higher concentration of rhNRGβ2. This effect on p21^(CIP1)expression was independent of the cell culture conditions used, sincesimilar effects were observed with both serum-free and serum-containingculture medium. Enhanced p21^(CIP1) expression with 10⁻⁸M rhNRGβ2 wassustained for at least 24 hours (a 48 hour incubation of cells withrhNRGβ2 results in an identical expression of p21^(CIP1)), and thus, maybe critical for the inhibition of DNA synthesis in cardiac muscle cellstreated with the high concentration of NRG. As shown in FIG. 5 b, IGF-Idid not stimulate p21^(CIP1) expression. To evaluate if the p21^(CIP1)response involves MAP kinase activation, cardiomyocytes were treatedwith the specific MAP kinase kinase (MEK1) inhibitor (PD98059). Both inthe presence or absence of serum, PD98059 blocked the increase inp21^(CIP1) expression induced by 10⁻⁸M rhNRGβ2 (FIG. 5 c), as well asthe increase in p42/44 MAP kinase phosphorylation (FIG. 5 d).

NRG Sarcomeric Structure and MHC Expression

To examine if NRG also affects embryonic myocardial cell structure andfunction, the effects of NRG on cardiomyocyte cytoskeletal andsarcomeric structures were evaluated. As shown in FIG. 6 a, rhNRGβ2(10⁻⁸M) stimulated both sarcomeric actin reorganisation (phalloidinstaining) and cardiac contractile unit assembly (staining of α-actininin Z-bands). In contrast, effects of 10⁻¹⁰M rhNRGβ2 were much lessevident (FIG. 6 a). A role for NRG in the regulation of myocardial cellfunction was also evident by the observation that rhNRGβ2 enhancedexpression of sarcomeric myosin heavy chains, while sarcomeric actinexpression remained unchanged (FIG. 6 b). Moreover, the effects ofrhNRGβ2 on cardiomyocytes were also sensitive to MEK1 inhibition byPD98059.

Discussion

Evidence provided indicates that ligand (NRG) concentration is a animportant factor in determining either the transient or sustainedactivation states of MAP kinases. The latter results in increasedexpression level of the Cdk inhibitor, p21^(CIP1), and is associatedwith decreased DNA synthesis in embryonic myocardial cells. This findingprovides clear support that the ligand gradient may decide cell fate incell differentiation and embryo development, and further furnishesmolecular insights on how intracellular signalling pathways distinguishthe signal strength based on ligand concentrations.

The importance of ligand concentration in cell differentiation has beensuspected for some time based on the following observations:

-   i) embryo developmental patterning is associated with a ligand    gradient;-   ii) ligand concentration is critical for cell differentiation in    vitro, and-   iii) overexpression of receptors in cells changes their fate in    response to ligand stimulation.

Taking these observations into consideration, NRGconcentration-dependent MAP kinase activation in embryonic myocardialcells establishes a model for further delineating the mechanisms of erbBreceptor-coupled cell signalling in reaction to changes in ligandconcentration.

The notion that NRG is a myocardial cell differentiation factor issupported by the finding that NRG induces expression of p21^(CIP1) inembryonic myocardial cells. As p21^(CIP1) is well documented to be aninhibitor of Cdk, which promotes entry from the G1 to the S phase of thecell cycle, increased expression of this protein in myocardial cellscould be critical for the initiation of terminal differentiation. Thisis also supported by previous findings that p21^(CIP1) expressionincreases in vivo with the onset of myocardial cell terminaldifferentiation (Parker et al. (1995) Science 267:1024–1027), as well aswith skeletal muscle cell differentiation (Dias et al. (1994) Semin.Diagn Pathol. 11:3–14). In the latter process, increased p21^(CIP1)expression eventually results in an exit from the cell cycle anddifferentiation. Since increase in p21^(CIP1) expression occurs prior tothat of other cell cycle regulators, it is used as an early marker forskeletal muscle differentiation. As demonstrated here, expression ofp21^(CIP1) is concomitant with the decrease in DNA synthesis inNRG-stimulated myocardial cells, suggesting the physiological role ofNRG-stimulated p21^(CIP1) expression in these cells. Furthermore, theinhibition of both MAP kinases and p21^(CIP1) by the ERK kinasesinhibitor assessed that NRG-stimulated p21^(CIP1) expression is a directresult of activation of MAP kinases.

The sustained activation of MAP kinases is required for induction ofp21^(CIP1) constitutive expression in cultured myocardial cells, whereastransient MAP kinase activation results in temporal expression ofp21^(CIP1). The latter is presumably insufficient to regulate the Cdkactivity, since p21^(CIP1) will be quickly degraded and constitutiveexpression is essential for blocking the cyclin/Cdk complex. In PC12cells, sustained activation of the MAP kinase pathway is confined to aresponse to specific signals from NGF receptors. The sustainedactivation of MAP kinases causes PC12 cell differentiation becomingneuronal cells. This pathway in cardiac myocytes, however, is able todifferentially respond to NRG concentration-based signal strength.

Further evidence support that NRG is a differentiation factor is thatNRG stimulate assembly of sarcomeric and cytoskeleton structures, whichoccur as myocardial progenitor cells differentiate to cardiac musclecells. Previous observation also indicated that more differentiatedcells have more organised sarcomeres (Rumynatsev, P.P. (1977) inInternational Review Cytology 51, pp 187–273). In a comparison of cellsstimulated with either PE or IGF-1, NRG-stimulated cells have the bestorganised sarcomeres. More importantly, when NRG is mixed with PE orIGF-1, NRG greatly improved sarcomeres, indicating that NRG is dominantin stimulation of sarcomere assembly in presence of other cell signals.NRG overrides the PE-mediated negative regulation of MHC-a expression,indicating that NRG is involved in the maintenance of adult type ofcontractile proteins. As previous studies indicated that NRG, ErbB2 andErbB4 are expressed in adult heart, NRG should play a role in themaintenance of myocardial cell differentiation state.

Two very important features of heart failure associated withcardiomyopathy in patients are disarrays of myofibers and sarcomeres.The former is the loose of the cell-cell adhesion and the latter is theloose of the sarcomere organisation. These pathological conditionswidely exist from congestive heart failure to dilated cardiomyopathy andseverely affect heart function. Currently no treatment is target on theassembly of cell-cell adhesion and sacomere structures. NRG clearlyplays a role in the process of the assembly and maintenance of cell-celladhesion and sarcomeric structures. That NRG stimulates myocardial celldifferentiation and the assembly of sarcomeric structures indicates thatcardiac muscle cell differentiation is associated with its cellstructure remodelling. Such a conclusion is consistent with generalobservation from heart muscle cell differentiation during heartdevelopment: differentiated muscle cells always contain well organisedsarcomeres.

In summary, that NRG is a differentiation factor for myocardial cells issupported by following evidence:

-   i) NRG stimulates sustained activation of MAP kinases;-   ii) NRG enhances p21^(CIP1) expression;-   ii) NRG inhibits IGF-1-stimulated DNA synthesis; and-   iv) NRG stimulates the myocardial cell assembly of sarcomeric and    cytoskeleton structures.-   v) NRG stimulates expression of the adult-type MHC gene.    Therapeutic Use

The present invention provides methods for treating or preventing heartfailure or cardiac muscle cell hypertrophy in a mammal by providing aneffective amount of a neuregulin. Preferably, the mammal is a humanpatient suffering from or at risk of developing heart failure.

The present invention is useful in preventing heart failure andcardiomyopathy in patients being treated with a drug which cause cardiachypertrophy or congestive heart failure, e.g., fludrocortisone acetateor herceptin. In the method of the invention, a neuregulin polypeptidecan be given prior to, simultaneously with, or subsequent to a drugwhich causes cardiac diseases.

In the therapeutic method of the invention, a neuregulin polypeptide isadministered to a human patient chronically or acutely, for example byinjection into the patient's vein. Optionally, neuregulin isadministered chronically in combination with an effective amount of acompound that acts to suppress a different hypertrophy induction pathwaythan a neuregulin Additional optional components include a cardiotrophicinhibitor such as a Ct-1 antagonist, an ACE inhibitor, such ascaptopril, and/or human growth hormone and/or IGF-I in the case ofcongestive heart failure, or with another anti-hypertrophic,myocardiotrophic factor, anti-arrhythmic, or inotropic factor in thecase of other types of heart failure or cardiac disorder.

The present invention can be combined with current therapeuticapproaches for treatment of heart failure, e.g., with ACE inhibitortreatment. ACE inhibitors are angiotensin-converting enzyme inhibitingdrugs which prevent the conversion of angiotensin I to angiotensin II.The ACE inhibitors may be beneficial in congestive heart failure byreducing systemic vascular resistance and relieving circulatorycongestion. ACE inhibitors include drugs designated by the trademarksAccupril® (quinapril), Altace® (ramipril), Capoten® (captopril),Lotensin® (benazepril), Monopril® (fosinopril), Prinivil® (lisinopril),Vasotec® (enalapril), and Zestril® (lisinopril).

The present invention can be combined with the administration of drugtherapies for the treatment of heart diseases such as hypertension. Forexample, a neuregulin polypeptide can be administered with endothelinreceptor antagonists, for example, and antibody to the endothelinreceptor, and peptide or other such small molecule antagonists;O-adrenoreceptor antagonists such as carvedilol; α₁-adrenoreceptorantagonists; anti-oxidants; compounds having multiple activities (e.g.,β-blocker/α-blocker/anti-oxidant); carvedilol-like compounds orcombinations of compounds providing multiple functions found incarvedilol; growth hormone, etc.

Neuregulin agonists alone or in combination with other hypertrophysuppressor pathway agonists or with molecules that antagonise knownhypertrophy induction pathways, are useful as drugs for in vivotreatment of mammals experiencing heart failure, so as to prevent orlessen heart failure effects.

Therapeutic formulations of agonist(s) for treating heart disorders areprepared for storage by mixing the agonist(s) having the desired degreeof purity with optional physiologically acceptable carriers, excipients,or stabilisers (Remington's Pharmaceutical Sciences, 16^(th) edition,Oslo, A., Ed., 1980), in the form of lyophilised cake or aqueoussolutions. Acceptable carriers, excipients, or stabilisers are non-toxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counter ions such as sodium; and/or non-ionic surfactantssuch as Tween, Pluronics, or polyethylene glycol (PEG). Theantagonist(s) are also suitably linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Theamount of carrier used in a formulation may range from about 1 to 99%,preferably from about 80 to 99%, optimally between 90 and 99% by weight.

The agonist(s) to be used for in vivo administration should be sterile.This is readily accomplished by methods known in the art, for example,by filtration through sterile filtration membranes, prior to orfollowing lyophilisation and reconstitution. The agonist(s) ordinarilywill be stored in lyophilised form or in solution.

Therapeutic agonist compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.The agonist(s) administration is in a chronic fashion only, for example,one of the following routes: injection or infusion by intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional routes, orally or usingsustained-release systems as noted below. Agonist(s) are administeredcontinuously by infusion or by periodic bolus injection if the clearancerate is sufficiently slow, or by administration into the blood stream orlymph. The preferred administration mode is targeted to the heart, so asto direct the molecule to the source and minimise side-effects of theagonists.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in form of shaped articles, e.g., films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al. (1981) J. Biomed. Mater. Res. 15: 167–277 andLanger (1982) Chem. Tech. 12: 98–105, or poly(vinyl alcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al. (1983)Biopolymers 22: 547–556), non-degradable ethylene-vinyl acetate (Langeret al. (1981) supra) degradable lactic acid-glycolic acid copolymerssuch as the Lupron Depot™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

The agonist(s) also may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerisation(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-[methylmethacylate] microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease molecules for shorter time periods. When encapsulated moleculesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilisation depending on the mechanisminvolved, e.g., using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release agonist(s) compositions also include liposomallyentrapped agonists(s). Liposomes containing agonists(s) are prepared bymethods known per se: DE 3.218,121; Epstein et al. (1985) Proc. Natl.Acad. Sci. USA 82: 3688–3692; Hwang et al. (1980) Proc. Natl. Acad. Sci.USA 77: 4030–4034; EP 52.322: EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application 83–118008; U.S. Pat. Nos. 4,485,045and 4,544.545; and EP 102, 324. Ordinarily the liposomes are of thesmall (about 200–800 Å) unilamellar type in which the lipid content isgreater than about 30 mol % cholesterol, the selected proportion beingadjusted for the optimal agonist therapy. A specific example of suitablesustained-release formulation is in EP 647,449.

An effective amount of NRG to be employed therapeutically will depend,for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient Accordingly, it willusually be necessary for the clinician to titre the dosage and modifythe route of administration as required to obtain the optimaltherapeutic effect.

NRG optionally is combined with or administered in concert with otheragents for treating congestive heart failure, including ACE inhibitors,CT-1 inhibitors, human growth hormone, and/or IGF-I. The effectiveamounts of such agents, if employed, will be at the clinician'sdiscretion. Dosage administration and adjustment are determined bymethods known to those skilled in the art to achieve the best managementof congestive heart failure and ideally takes into account use ofdiuretics or digitalis, and conditions such as hypotension and renalimpairment. The dose will additionally depend on such factors as thetype of drug used and the specific patient being treated. Typically theamount employed will be the same dose as that used if the drug were tobe administered without agonist; however, lower doses may be employeddepending on such factors as the presence of side-effects, the conditionbeing treated, the type of patient, and the type of agonists and drug,provided the total amount of agents provides an effective dose for thecondition being treated.

Thus, for example, in the case of ACE inhibitors, a test dose ofenalapril is 5 mg, which is then increased up to 10–20 mg per day, oncea day, as the patient tolerates it. As another example, captopril isinitially administered orally to human patients in a test dose of 6.25mg and the dose is then escalated, as the patient tolerates it to 25 mgtwice per day (BID) or three times per day (TID) and may be titrated to50 mg BID or TID. Tolerance level is estimated by determining whetherdecrease in blood pressure is accompanied by signs of hypotension. Ifindicated, the dose may be increased up to 100 mg BID or TID. Captoprilis produced for administration as the active ingredient, in combinationwith hydrochlorothiazide, and as a pH stabilised core having an entericor delayed release coating which protects captopril until it reaches thecolon. Captopril is available for administration in tablet or capsuleform. A discussion of the dosage. Administration, indications andcontraindications associated with captopril and other ACE inhibitors canbe found in the Physicians Desk Reference, Medical Economics DataProduction Co., Montvale, NJ. 2314–2320 (1994).

In an example of an injectable therapeutic composition of neuregulin,the formulation contains 1% neuregulin and 99% saline, where neuregulinis a polypeptide thereof. In another example of an injectabletherapeutic composition of neuregulin, the formulation contains 5% ofthe neuregulin polypeptide, 1% ACE inhibitor captopril, and 94% saline.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1. A method of inducing remodeling of cardiac muscle cell sarcomeric andcytoskeleton structures or cell-cell adhesions, which method comprisescontacting cardiac muscle cells with a neuregulin protein consisting ofthe amino acid sequence set forth in SEQ ID NO:2, in the amountsufficient to activate the MAP kinase pathway in said cardiac musclecells and induce remodeling of said cardiac muscle cell sarcomeric andcytoskeleton structures or cell-cell adhesions.
 2. The method of claim1, wherein the neuregulin protein is used in an amount that is at least10⁻⁸M.
 3. The method of claim 1, wherein the cardiomyocyte or thecardiac muscle cells exist in a mammal.
 4. The method of claim 3,wherein the mammal is a human.
 5. The method of claim 4, wherein thehuman has or is suspected of having a heart failure.
 6. The method ofclaim 5, wherein the heart failure is a disease state selected from thegroup consisting of congestive heart failure, myocardial infarction,tachyarrhythmia, familial hypertrophic cardiomyopathy, ischaemic heartdisease, idiopathic dilated cardiomyopathy and myocarditis.
 7. Themethod of claim 5, wherein the heart failure is in the form ofischaemic, congenital, rheumatic, or idiopathic.
 8. The method of claim5, wherein the heart failure results from disassociation of cardiacmuscle cell-cell adhesion and/or the disarray of sarcomeric structuresin the mammal.
 9. The method of claim 3, wherein the neuregulin proteinis administered with a pharmaceutically acceptable carrier or excipient.10. The method of claim 1, wherein the contact of the cardiac musclecells with the neuregulin protein decreases DNA synthesis in the cardiacmuscle cells.
 11. The method of claim 1, wherein the contact of thecardiac muscle cells with the neuregulin protein results in sustainedactivation of the MAP kinase pathway in the cardiac muscle cells. 12.The method of claim 3, wherein the neuregulin protein is administeredorally, using a sustained-release system or via injection or infusion.13. The method of claim 12, wherein the injection or infusion isselected from the group consisting of intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial andintralesional injection or infusion.
 14. The method of claim 1, whichfurther comprises contacting the cardiac muscle cells with an effectiveamount of an agent which causes cardiac hypertrophy or congestive heartfailure.
 15. The method of claim 14, wherein the agent which causescardiac hypertrophy or congestive heart failure is fludrocortisoneacetate or herceptin.
 16. The method of claim 1, which further comprisescontacting the cardiac muscle cells with an effective amount of an agentthat acts to suppress a hypertrophy induction pathway different from thepathway suppressed by the neuregulin.
 17. The method of claim 16,wherein the agent that acts to suppress a hypertrophy induction pathwaydifferent from the pathway suppressed by the neuregulin is selected fromthe group consisting of a cardiotrophic inhibitor, anangiotensin-converting enzyme (ACE) inhibitor, a human growth hormone,an IGF-I, an anti-hypertrophic, myocardiotrophic factor, ananti-arrhythmic factor and an inotropic factor.
 18. The method of claim1, which further comprises contacting the cardiac muscle cells with aneffective amount of an angiotensin-converting enzyme (ACE) inhibitor.19. The method of claim 18, wherein the ACE inhibitor is selected fromthe group consisting of quinapril, ramipril, captopril, benazepril,fosinopril, lisinopril, enalapril and lisinopril.
 20. The method ofclaim 1, which further comprises contacting the cardiac muscle cellswith an effective amount of an agent for treating hypertension.
 21. Themethod of claim 20, wherein the agent for treating hypertension isselected from the group consisting of an antibody to the endothelinreceptor, a β-adrenoreceptor antagonist, an α₁-noreceptor antagonist, ananti-oxidant, a β-blocker and a growth hormone.
 22. A method fortreating or delaying disassociation of cardiac muscle cell-cell adhesionand/or the disarray of sarcomeric structures in a mammal, which methodcomprises administering to a mammal, to which such treatment or delay isneeded or desirable, a neuregulin protein consisting of the amino acidsequence set forth in SEQ ID NO:2, in an amount sufficient to activatethe MAP kinase pathway in said mammal, whereby said disassociation ofcardiac muscle cell-cell adhesion and/or the disarray of sarcomericstructures is treated or delayed in said mammal.
 23. The method of claim22, wherein the mammal is a human.
 24. The method of claim 23, whereinthe human has or is suspected of having a heart failure.
 25. The methodof claim 24, wherein the heart failure is a disease state selected fromthe group consisting of congestive heart failure, myocardial infarction,tachyarrhythmia, familial hypertrophic cardiomyopathy, ischaemic heartdisease, idiopathic dilated cardiomyopathy and myocarditis.
 26. Themethod of claim 24, wherein the heart failure is in the form ofischaemic, congenital, rheumatic, or idiopathic.
 27. The method of claim22, wherein administration of the neuregulin protein decreases DNAsynthesis in the cardiac muscle cells of the mammal.
 28. The method ofclaim 22, wherein administration of the neuregulin protein results insustained activation of the MAP kinase pathway in the cardiac musclecells of the mammal.